Gastrointestinal Physiology - Gianrico Farrugia

Introductory Work in Mechanosensitive Calcium and Potassium Channels

[effect of increasing intracellular pressure on Ca2+ current]

Figure 1: Activation of inward Ca2+ current by increased intracellular pressure. Peak inward current at atmospheric pressure measured 48 pA (a). Increase in intracellular pressure to 10 mmHg above atmospheric increased peak inward Ca2+ current to 110 pA (b). No change occurred in the voltage at which peak inward current was measured (c).


[effect of varying increases in intracellular pressure on Ca2+ current]

Figure 2: Increase in inward current evoked by sequential increases in intracellular pressure. Peak inward Ca2+ current (a) was -26 pA at atmospheric pressure, -37 pA at 10 mmHg above atmospheric pressure, -51 pA at 20 mmHg and -16 pA at 20 mmHg in the presence of nifedipine (1 µM).


[percentage increase in Ca2+ current upon exposure to perfusion]

Figure 3: Percentage increase in calcium current for single cells upon perfusion at 10ml/min. Mean increase in current is 29.2%.


[effect of varying rates of perfusion on Ca2+ current]

Figure 4: Activation of inward Ca2+ current by external shear force (a). Peak control inward Ca2+ current at a bath flow rate of 0 ml/min measured 66 pA. Inward current increased to 70 pA at a bath flow rate of 5 ml/min, 78 pA at 10 ml/min, and 97 pA at 20 ml/min. Peak inward current returned to baseline 6 min after stopping bath perfusion (b). Numbers indicate flow rate (ml/min).


[effect of pressure on Ca2+ current at the single channel level]

Figure 5: Single channel recordings obtained from an on-cell patch with 80 mM barium as the charge carrier in the pipette. Little channel activity was noted at atmospheric pipette pressure (NPo = 0.005, a). Negative pressure (-40 mmHg) activated a nifedipine-sensitive channel (NPo = 0.06, b and NPo=0.0004, c).


[effect of bath perfusion at 10 ml/min on outward K+ current]

Figure 6: Effect of bath perfusion at 10 ml/min on outward K+ current. Whole cell currents were recorded with K+ in the pipette to record outward current and membrane potential (A) using the pulse protocol shown in the inset. Bath perfusion increased outward K+ current from 680 pA to 841 pA at +60 mV (B). 4 min after bath perfusion outward current (at +60 mV) decreased to 719 pA (C). Bath perfusion hyperpolarized the membrane potential from -52 to -64 mV. Membrane potential depolarized back to -53 mV 3 min after perfusion (circle=control, square=perfusion, triangle=4 min post perfusion [D]). The time course of the changes in membrane potential is shown in E (bar = perfusion). The mean change in outward K+ current (inset) was from 664 plus or minus 57 pA to 773 plus or minus 72 pA (n = 14, * P < 0.0002, red=control, green=perfused). The membrane potential hyperpolarized from ?42 plus or minus 4 mV to ?50 plus or minus 5 mV (+P < 0.01, red=control, green=perfused).


[effect of bath perfusion at 10 ml/min on inward Ca2+ current and outward K+ current]

Figure 7: Effect of bath perfusion at 10 ml/min on inward Ca2+ current and outward K+ current. Currents were recorded using the pulse protocol shown in the inset. In 50% of cells recorded from with K+ in the pipette an inward Ca2+ current was discernible (A). Bath perfusion (B) increased inward Ca2+ current in this cell from 17 pA to 31 pA (insets, scale same for both A and B). Outward current increased from 480 pA to 525 pA in the recording shown, obtained 30 sec after initiation of perfusion, and peaked 90 sec after initiation of perfusion.


[effect of block of the mechano-sensitive Ca2+ channel by nifedipine (10 uM) in bath on outward K+ current]

Figure 8: Effect of block of the mechano-sensitive Ca2+ channel by nifedipine (10 uM) in bath on outward K+ current. Currents were recorded using the pulse protocol shown in the inset. In the presence of nifedipine (A), perfusion (B) did not increase outward K+ current (1465pA to 1488pA at +60 mV) and (C) did not change membrane potential (-44 to -43 mV, circle=control in the presence of nifedipine, square=perfusion). The mean change in outward current (inset) was from 694 plus or minus 133 to 713 plus or minus 132 (n = 9, P > 0.05, red=control, green=perfusion with nifedipine) and the mean change in membrane potential was from -47 plus or minus 5 mV to ?48 plus or minus 6 mV (P > 0.05, red=control, green=perfusion with nifedipine).


[effect of block of large conductance Ca2+-activated K+ channels on the perfusion-induced increase in outward current]

Figure 9: Effect of block of large conductance Ca2+-activated K+ channels on the perfusion-induced increase in outward current. Currents were recorded using the pulse protocol shown in the inset. Addition of charybdotoxin (100 nM) to the bath decreased outward current (A, B). In the presence of charybdotoxin, perfusion of the bath (C) did not increase outward current (573 to 561 pA) or (D) change membrane potential (-38 to -39 mV, red=control, square=charybdotoxin, triangle=perfusion in the presence of charybdotoxin). The mean change in outward current (inset), in the presence of charybdotoxin, was from 550 plus or minus 32 pA to 518 plus or minus 49 pA (red=control, green=perfusion with charybdotoxin) and the mean change in membrane potential was from -39.3 plus or minus 2 mV to -42.0 plus or minus 2 mV (n = 7, P > 0.05, red=control, green=perfusion with charybdotoxin).


[effect of perfusion on large conductance Ca2+-activated K+ channel open probability]

Figure 10: Effect of perfusion on large conductance Ca2+-activated K+ channel open probability. Records were obtained from an on-cell patch with 150 K+ in pipette and normal Ringer?s solution in bath. Normal Ringer?s solution was perfused at 10 ml/min for 30 sec to activate mechano-sensitive L-type Ca2+ channels. 1 min records were obtained at a Vpipette of -40 mV immediately before and after perfusion. No voltage command was applied between records. NPo was 0.01 before and 0.08 after perfusion (O open, C closed). Single channel conductance in this patch was 93 plus or minus 5 pS.


[effect of perfusion on cell length]

Figure 11: Effect of perfusion on cell length. Panel A shows a human jejunal circular smooth muscle cell just before perfusion of normal Ringer?s solution, panel B during perfusion (10 ml/min for 30 sec), and panel C about 30 sec after perfusion was stopped. Size of the line is the same for each panel. Perfusion decreased cell length by 11% which reversed when perfusion was stopped.


[proposed model]

Figure 12: Proposed model. Mechano-activation of L-type Ca2+ channels in human jejunal circular smooth muscle cells (A, B) increases Ca2+ entry and activates the contractile apparatus (C). Ca2+ entry subsequently activates large conductance Ca2+-activated K+ channels (D) resulting in an increase in outward K+ current, membrane hyperpolarization (E) and relaxation (F).

Appointments | Contact Us | Give to Mayo Clinic | Search

LEGAL RESTRICTIONS AND TERMS OF USE APPLICABLE TO THIS SITE

Use of this site signifies your agreement to the terms of use.
© 2012 Mayo Foundation for Medical Education and Research. All rights reserved.